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PubMed1993

Alu and L1 sequence distributions in Xq24-q28 and their comparative utility in YAC contig assembly and verification.

The contents of Alu- and L1-containing TaqI restriction fragments were assessed by Southern blot analyses across YAC contigs already assembled by other means and localized within Xq24-q28. Fingerprinting patterns of YACs in contigs were concordant, and using software based on that of M. V. Olson et al. (1986, Proc. Natl. Acad. Sci. USA 83: 7826) to analyze digitized data on fragment sizes, fingerprinting itself could establish matches among about 40% of a test group of 435 YACs. At 100-kb resolution, both repetitive elements were found throughout the region, with no apparent enrichment of Alu or L1 in DNA of G compared to that found in R bands. However, consistent with a random overall distribution, delimited regions of up to 100 kb contained clusters of repetitive elements. The local concentrations may help to account for the reported differential hybridization of Alu and L1 probes to segments of metaphase chromosomes.

Chromosome Mapping↗

PubMed2006

Phylogenetic supermatrix analysis of GenBank sequences from 2228 papilionoid legumes.

A comprehensive phylogeny of papilionoid legumes was inferred from sequences of 2228 taxa in GenBank release 147. A semiautomated analysis pipeline was constructed to download, parse, assemble, align, combine, and build trees from a pool of 11,881 sequences. Initial steps included all-against-all BLAST similarity searches coupled with assembly, using a novel strategy for building length-homogeneous primary sequence clusters. This was followed by a combination of global and local alignment protocols to build larger secondary clusters of locally aligned sequences, thus taking into account the dramatic differences in length of the heterogeneous coding and noncoding sequence data present in GenBank. Next, clusters were checked for the presence of duplicate genes and other potentially misleading sequences and examined for combinability with other clusters on the basis of taxon overlap. Finally, two supermatrices were constructed: a "sparse" matrix based on the primary clusters alone (1794 taxa x 53,977 characters), and a somewhat more "dense" matrix based on the secondary clusters (2228 taxa x 33,168 characters). Both matrices were very sparse, with 95% of their cells containing gaps or question marks. These were subjected to extensive heuristic parsimony analyses using deterministic and stochastic heuristics, including bootstrap analyses. A "reduced consensus" bootstrap analysis was also performed to detect cryptic signal in a subtree of the data set corresponding to a "backbone" phylogeny proposed in previous studies. Overall, the dense supermatrix appeared to provide much more satisfying results, indicated by better resolution of the bootstrap tree, excellent agreement with the backbone papilionoid tree in the reduced bootstrap consensus analysis, few problematic large polytomies in the strict consensus, and less fragmentation of conventionally recognized genera. Nevertheless, at lower taxonomic levels several problems were identified and diagnosed. A large number of methodological issues in supermatrix construction at this scale are discussed, including detection of annotation errors in GenBank sequences; the shortage of effective algorithms and software for local multiple sequence alignment; the difficulty of overcoming effects of fragmentation of data into nearly disjoint blocks in sparse supermatrices; and the lack of informative tools to assess confidence limits in very large trees.

Algorithms↗

PubMed2003

Collaborative consensus for optimized multilocus sequence typing of Candida albicans.

A panel of 86 different Candida albicans isolates was subjected to multilocus sequence typing (MLST) in two laboratories to obtain sequence data for 10 published housekeeping gene fragments. Analysis of data for all possible combinations of five, six, seven, eight, and nine of the fragments showed that a set comprising the fragments AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b was the smallest that yielded 86 unique diploid sequence types for the 86 isolates. This set is recommended for future MLST with C. albicans.

Candida albicans↗

PubMed1994

Human gallstones contain pronucleating nonmucin glycoproteins that are immunoglobulins.

OBJECTIVE: Pronucleating nonmucin glycoproteins in human cholesterol and black gallstones were isolated and identified. SUMMARY BACKGROUND DATA: Gallbladder bile contains nonmucin glycoproteins that are pronucleating of cholesterol monohydrate crystals. Little is known about the presence or activity of these proteins within gallstones. METHODS: Nonmucin glycoproteins were isolated from single cholesterol (n = 8), multiple cholesterol (n = 8), and black pigment (n = 8) gallstones by concanavalin A lectin-affinity chromatography. The proteins were separated by sodium dodecyl sulfate gradient electrophoresis. Western blot analysis was performed for Fab immunoglobulin fragments, and heavy chains from the immunoglobulin G, A, E, and M subclasses. A crystal observation time assay was performed on the combination of isolated nonmucin glycoproteins from gallstones and isolated Fab fragments. RESULTS: Nonmucin glycoproteins of molecular weights 10, 15, 17, 22, 28, and 208 kD were identified in gallstones. These six nonmucin glycoproteins shortened the crystal observation time by more than 50% (p < 0.01) compared with model bile. Western blot analysis confirmed the identity of the 22- and 28-kD proteins as immunoglobulin Fab fragments. These were seen in all gallstones, irrespective of the gallstone type. The isolated Fab 28-kD fragment from the gallstones of 23 patients shortened the extrapolated crystal observation time by 78% (p < 0.01). However, commercially available Fab fragments had no effect on either cholesterol crystal appearance or growth. CONCLUSIONS: Nonmucin glycoproteins that are pronucleating for cholesterol monohydrate crystals are also found in human cholesterol and black pigment gallstones. Fab immunoglobulin fragments were found in all gallstones irrespective of the gallstone type. Fab immunoglobulin fragments from gallstones shortened the crystal observation time but not crystal growth or total crystal content compared with model bile or commercially available Fab fragments. These data suggest that an antigen-immune (Fab) complex may contribute to cholesterol crystal function.

Bile Pigments↗

PubMed1986

Is counting the dead enough? Strategies for monitoring health needs in New York City.

Determining health care needs--especially unmet needs--in New York City should continue as a research and policy priority, especially in a rapidly changing health care environment. Among the many data sources that can be used in assessing needs are: mortality data; census data; other vital statistics; National Health Interview Survey data; other interview and survey data; physician examination of patients. Among the uses to which these and other data are being put in an effort to determine unmet need and health status are: predicting age-sex-specific demographic, health status, and utilization trends; analysis of sentinel health events; developing hierarchies of need for small areas; development of city and neighborhood "health profiles"; provider determination of needs for program planning. Obstacles to progress persist, however, including the increasing costs of data gathering and analysis; lack of popular or political support for needs assessment; and problems in defining what health care needs are. Among high priorities for those interested in better assessment of health care needs in New York City are: recreating public and political interest in the issue; garnering more financial support for research; countering trends toward privatization and fragmentation of data collection and analysis efforts; new relationships between academic and other researchers; better definition of need and refinement of research methodologies and data bases; development of a clear strategy for assessing health needs in New York City in the future, and garnering broad support for implementing it.

Data Collection↗

PubMed1987

Ribosomal DNA sequences attached to the nuclear matrix.

The organization of rat liver ribosomal DNA (rDNA) as matrix-attached DNA loops was examined using a protocol which fractionates chromatin from discrete regions of DNA loops. Southern blot analysis of matrix-attached and solubilized chromatin DNA fragments demonstrated that rDNA is associated with the matrix via its 5' and 3' nontranscribed spacer sequences (NTS). Although the 45 S rRNA coding sequences were approximately threefold enriched in matrix preparations, the recovery of this DNA (unlike the NTS) was dependent on the extent of nuclease digest and proportional to the length of the matrix-attached DNA fragments. The data suggest that rDNA is organized as matrix-attached DNA loops and only the NTS are directly involved in matrix binding. Further, we demonstrated that while the kinetics and extent of nuclease digestion were similar in all regions of the DNA loops, the nuclease digestion pattern of bulk nuclear and matrix DNA showed a typical nucleosome organization, but the rDNA fragments retained with the nuclear matrix did not.

Animals↗

PubMed2006

Isolation and characterization of a pseudomonas oleovorans degrading the chloroacetamide herbicide acetochlor.

To date, no pure bacterial cultures that could degrade acetochlor have been described. In this study, one strain of microorganism capable of degrading acetochlor, designated as LCa2, was isolated from acetochlor-contaminated soil. The strain LCa2 is Pseudomonas oleovorans according to the criteria of Bergey's manual of determinative bacteriology and sequence analysis of the partial 16S rRNA gene. Optimum growth temperature and pH were 35 degrees C and 8.0, respectively. The strain could degrade 98.03% of acetochlor treated at a concentration of 7.6 mg l(-1) after 7 days of incubation and could tolerate 200 mg l(-1) of acetochlor. When the acetochlor concentration became higher, the degradation cycle became longer. The acetochlor biodegradation products were identified by GC-MS based on mass spectral data and fragmentation patterns. The main plausible degradative pathways involved dechlorination, hydroxylation, N-dealkylation, C-dealkylation and dehydrogenation.

Herbicides↗

PubMed2000

Nuclear translocation of granzyme B in target cell apoptosis.

Granzyme B is the prototypic member of a family of serine proteases localized to the cytolytic granules of cytotoxic lymphocytes. Together with another granule protein, perforin, granzyme B is capable of inducing all aspects of apoptotic death in target cells. A number of granzyme B substrates have been identified and it has been demonstrated that granzyme B is responsible, directly or indirectly, for the morphological nuclear changes observed in target cell apoptosis, including DNA fragmentation. In an earlier study, we showed that granzyme B binds to a nuclear protein in a manner dependent on its enzymatic activity. Here, we demonstrate that granzyme B is translocated rapidly to the nucleus in cells that have been induced to undergo apoptosis by a granzyme-dependent process, and that translocation is dependent on caspase activity. Appearance of granzyme B in the nucleus of target cells precedes the detection of DNA fragmentation. Although not directly responsible for DNA fragmentation, these data suggest a nuclear role for granzyme B in target cell apoptosis. c-Abl nuclear functions.

Animals↗

PubMed2002

Inhibition of prolactin secretion from the male rat anterior pituitary by cryptic sequences of prothyrotropin releasing hormone, ProTRH178-199 and ProTRH186-199.

Previous studies have shown that intronic peptide sequences in the prohormone for thyrotropin-releasing hormone (TRH) have physiological actions on pituitary hormone secretion. The aim of this investigation was to examine the effect of the cryptic peptides, prothyrotropin- releasing hormone(178-199) (ProTRH(178-199)) and ProTRH(186-199), on prolactin (PRL) release from the anterior pituitary. Perifusion studies were performed with anterior pituitaries obtained from individual adult male Sprague Dawley rats at 70 90 d of age. Perifusate was collected in 5-min fractions for 25 min prior to peptide administration and for 60 min afterward. Pituitaries were perifused with a single 5 min pulse of either 2, 10, or 40 nM concentrations (peak pulse) of each peptide or the vehicle. Sixty minutes after peptide administration, a 200 mM pulse of potassium chloride was delivered to check tissue viability. Prolactin was measured in the perifusate by radioimmunoassay. Results showed that both peptides induced a significant long-term suppression of prolactin secretion that was still evident at 60 min after peptide exposure. ProTRH(186-199) was similar to ProTRH(178-199) in suppressing prolactin release at the 2 and 40 nM dose, suggesting that the amino acid sequence necessary for prolactin inhibition is contained within the smaller peptide fragment. These data indicate that a cryptic sequence within the proTRH peptide can have biological activity at the level of the anterior pituitary gland in regulating prolactin secretion.

Animals↗

PubMed1996

Role of endonuclease activity and DNA fragmentation in Ca2+ ionophore A23187-mediated injury to rabbit isolated gastric mucosal cells.

In the current study, the role of endonuclease activity in calcium ionophore A23187-induced gastric mucosal cellular disruption was examined using rabbit gastric mucosal cells. Cell integrity was assessed using trypan blue dye exclusion and Alamar blue dye absorbance. Ionophore A23187 (1.6-25 microM) induced a concentration-dependent decrease in dye exclusion and cell metabolism in cells suspended in a medium containing Ca2+ (2 mM), while no such effect was observed in cells incubated in the absence of extracellular Ca2+. Cells that were pretreated with the endonuclease inhibitors aurintricarboxylic acid (ATCA; 0.2 or 0.5 mM or Zn2+; 0.01 and 0.1 mM) exhibited significant reduction in the total extent of cell injury when incubated with A23187 in the presence of Ca2+. DNA fragmentation as assessed by measurement of [3H]thymidine liberation or gel electrophoresis was increased in response to ionophore A23187 (12.5 or 25 microM) treatment. A minimal degree of fragmentation was observed when cells were suspended in a Ca(2+)-free medium or incubated in the presence of ATCA or Zn2+. Addition of ethanol (8% w/v) induced a significant increase in cell injury, which was not affected by either removal of extracellular Ca2+ or ATCA pretreatment. Furthermore, treatment with the antioxidants catalase (50 micrograms/ml) or 2',2'-dipyridyl (2 mM) reduced ionophore-induced cell injury but did not reduce the extent of DNA fragmentation. These data suggest that sustained increases in intracellular Ca2+ result in increased endonuclease activity in gastric mucosal cells, leading to extensive DNA lysis and cell damage. Ethanol-induced cell damage does not involve Ca2+ influx and therefore is not mediated by endonuclease activation. Furthermore, sustained increases in cellular Ca2+ may also mediate their effects via formation of reactive oxygen metabolites, but this mechanism of cell damage does not appear to involve DNA fragmentation.

Animals↗

PubMed1999

Thapsigargin enhances camptothecin-induced apoptosis in cardiomyocytes.

Topoisomerase I inhibitors are promising new chemotherapeutic agents for the treatment of certain malignancies. The present study investigated the impact of the topoisomerase I inhibitor camptothecin on cell death in cardiomyocytes and sought to determine whether the sesquiterpene gamma-lactone--thapsigargin, that alter sarcoplasmic reticulum calcium flux, modulates the effect of camptothecin on the cardiomyocyte. Camptothecin-induced cell death was demonstrated in cardiomyocytes maintained in culture, from 7 day old embryonic chick hearts, by the trypan blue and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, two independent indicators of the loss of cell viability. The type of cell death was attributed to apoptosis based on cell structure, DNA fragmentation and flow cytometry studies. Camptothecin-treated cardiomyocytes were shrunken with membrane blebs and nuclear fragmentation. Camptothecin produced a dose-dependent increase in DNA fragments of 180 base pairs, or multiples thereof, which are characteristic of apoptosis. A two-fold increase in this type of DNA fragmentation was produced by camptothecin (10 microM) compared to control (diluent-treated) cells. Flow cytometry analysis of populations of 10,000 cardiomyocytes stained with propidium iodide demonstrated a significant increase in the proportion of the population with alterations of DNA content consistent with apoptosis. Pretreatment of cells with thapsigargin, which selectively inhibits sarcoplasmic reticulum and endoplasmic reticulum Ca+2-dependent ATPase, significantly augmented camptothecin-induced apoptosis. Exploring further the role of calcium in camptothecin-induced cell death, we found that the Ca+2 chelator EGTA decreased camptothecin-induced DNA fragmentation. These data indicate the potential for cardiotoxicity from camptothecin through the process of apoptosis and suggest that agents which affect cellular calcium regulation enhance camptothecin-induced apoptosis.

Animals↗

PubMed2000

[Study of solid-phase catalytic isotopic exchange of hydrogen in alpha-conotoxin G1 under the effect of spillover-tritium].

Tritium-labeled alpha-conotoxin G1 with a molar radioactivity of 35 Ci/mmol and full biological activity (according to the binding to nicotinic acetylcholine receptor) was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE). The tritium distribution in the molecule of alpha-conotoxin G1 was revealed by 3H NMR spectroscopy. Tritium was found in all amino acid residues except for the Asn4-Pro5-Ala6 fragment. The data on the comparative reactivity of C-H bonds, the ab initio quantum-chemical calculation of the hydrogen exchange reaction, and the information on the spatial structures of alpha-conotoxin G1 in solution and in crystal state allowed us to establish that the reactivity of H atoms may be increased by their interaction with the electron donor O and N atoms at the transition state of the HSCIE reaction. A decrease in the rate of the HSCIE reaction could be caused by both a poor spatial accessibility of C-H bonds and a limited mobility of the peptide fragment containing these bonds.

Amino Acid Sequence↗

PubMed2003

Mitochondrial and endoplasmic reticulum stress-induced apoptotic pathways are activated by 5-aminolevulinic acid-based photodynamic therapy in HL60 leukemia cells.

We studied the mechanism of the cytotoxic effects of 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT; induction with 1 mM ALA for 4 h followed by a blue light dose of 18 J/cm(2)) on the human promyelocytic leukemia cell line HL60 using biochemical and electron microscopy methods. The disruption of mitochondrial membrane potential, deltapsi(m), was paralleled by a decrease in ATP level, unmasking of the mitochondrial antigen 7A6, release of cytochrome c into the cytoplasm, activation of caspases 9 and 3 and cleavage of poly(ADP-ribose) polymerase (PARP). This was followed by DNA fragmentation. These data suggest that ALA-PDT activates the mitochondrial apoptotic pathway. The level of endoplasmic reticulum Ca(2+)-binding chaperones ERp57 and ERp72 and of anti-apoptotic proteins Bcl-2 and Bcl-x(L) was decreased whereas that of Ca(2+)-binding protein calmodulin and the stress protein HSP60 was elevated following ALA-PDT. Inhibition of the initiator caspase 9, execution caspase 3 and Ca(2+)-dependent protease m-calpain, did not prevent DNA fragmentation. We conclude that, in our in vitro model, ALA-based photodynamic treatment initiates several signaling processes in HL60 cells that lead to rapidly progressing apoptosis, which is followed by slow necrosis. Two apoptotic processes proceed in parallel, one representing the mitochondrial pathway, the other involving disruption of calcium homeostasis and activation of the endoplasmic reticulum stress-mediated pathway.

Adenosine Triphosphate↗

PubMed1996

BCR-ABL does not prevent apoptotic death induced by human natural killer or lymphokine-activated killer cells.

The erythromyeloid cell line, K562, the most sensitive target in human natural killer (NK) cell mediated cytotoxicity, is derived from a chronic myeloid leukemia (CML) patient and expresses the characteristic reciprocal translocation t(9;22). The resulting BCR-ABL fusion protein has been shown to mediate the unusual resistance of K562, and other BCR-ABL expressing lines, to apoptosis induced by a variety of agents (irradiation, UV light, cytotoxic drugs). Here we show that human NK and lymphokine-activated killer (LAK) cells, when tested at low effector to target ratio, can readily induce apoptotic death in K562 cells. This was accompanied with classical DNA oligonucleosomal fragmentation, an unexpected finding given the reported lack of such fragmentation when apoptosis is induced in K562 by chemical agents, after downregulation of BCR-ABL. Apoptosis was assessed by several means: morphological studies, 125I-DNA versus 51Cr release, DNA agarose gel electrophoresis, and results were always concordant, with a delayed kinetics for DNA oligonucleosomal fragmentation. Similar data were obtained with a pluripotent human hematopoietic cell line, UT-7, infected with a defective amphotropic p210 BCR-ABL retrovirus. The BCR-ABL expressing subclone UT-7/9, while being no longer sensitive to cytotoxic drugs or to tumor necrosis factor, a lytic mediator to which UT-7 cells are sensitive, underwent apoptotic death when exposed to LAK effector cells to the same degree as the parental UT-7 line. With these targets, DNA oligonucleosomal fragmentation occurred concomitantly with isotope release. Results obtained with several inhibitors of exocytosis strongly suggest that cytotoxic granules mediate NK and LAK cell-induced apoptotic death. In conclusion, NK and LAK cell-induced apoptotic signals, unlike those activated by chemotherapeutic agents, are unaffected by the antiapoptotic action of BCR-ABL. This unique property may support the observed curative effect of allogeneic bone marrow transplantation in CML.

Apoptosis↗

PubMed2004

Involvement of vanilloid-like receptors in the effects of anandamide on motor behavior and nigrostriatal dopaminergic activity: in vivo and in vitro evidence.

The administration of the endocannabinoid anandamide to rats produces hypokinesia in parallel to a decrease in the activity of nigrostriatal dopaminergic neurons. It was earlier hypothesized that this effect was mediated through the activation of CB(1) receptors, although these receptors have not been found in dopaminergic neurons, but in striatal projection neurons connected with them. However, two recent discoveries: (i) that anandamide is also able to activate vanilloid VR(1) receptors, and (ii) that VR(1) receptors are located on nigrostriatal dopaminergic neurons, allow to re-evaluate this hypothesis and suggest that the activation of vanilloid-like receptors rather than CB(1) receptors might be responsible of anandamide-induced hypokinesia and decreased nigrostriatal dopaminergic activity. To validate this new hypothesis, we carried out two different experiments. First, we explored whether the inhibitory effects of anandamide on motor activity and dopaminergic transmission were reversed by capsazepine, an antagonist for vanilloid-like receptors. Our data demonstrated that anandamide reduced ambulation, stereotypies and exploration, measured in the open-field test, whereas it increased the time spent in inactivity. All these effects were completely reversed by capsazepine, which had no effect by itself. Anandamide also caused a significant decrease in nigrostriatal dopaminergic activity, reflected by a reduction in DOPAC contents in the caudate-putamen, which was also reversed by capsazepine. As a second objective, we explored whether anandamide is able to directly influence nigrostriatal dopaminergic function by examining its effects on in vitro dopamine (DA) release using perifused striatal fragments. Our data confirmed that anandamide significantly decreased K(+)-stimulated dopamine release from nigrostriatal terminals and that this effect was vanilloid-like receptor-mediated since it was prevented by capsazepine. This in vitro inhibitory effect was not seen with a classic cannabinoid agonist that does not bind vanilloid-like receptors. In summary, anandamide behaves as a hypokinetic substance, thus producing motor depression in the open-field test, presumably related to a decrease in nigrostriatal dopaminergic activity. These effects were completely reversed by the vanilloid-like receptor antagonist capsazepine, thus indicating a role of these receptors, which are located on dopaminergic neurons, in mediating hypokinetic effects of anandamide. In vitro studies, using perifused striatal fragments, support this vanilloid-like receptor-mediated direct action, which would not be available for classic cannabinoid agonists.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1995

Cytogenetic Effects Induced by Cytochalasin B in Chinese Hamster Ovary Cells.

We determined the kinetics of the induction of chromosomal aberrations and micronuclei (MN) by mitomycin C (MMC, 0.1 &mgr;g/ml) in Chinese hamster ovary (CHO) cells treated with cytochalasin B (Cyt-B, 3 &mgr;g/ml). In cells treated with Cyt-B as well as with Cyt-B plus MMC the highest yield of binucleated cells was obtained 24 h after treatment. After 40 h of treatment with Cyt-B the frequency of MN in binucleated cells was significantly higher than that observed at previous times in the same cultures as well as in controls. In cultures treated with MMC the frequency of MN increased with time, reaching the highest value at 24 h. The frequency of chromosomal aberrations was also significantly higher in cells treated both with Cyt-B and Cyt-B plus MMC than in controls and exceeded that of MN in parallel cultures. These data confirm the capacity of MMC to induce chromosomal alterations in mammalian cells; in particular they indicate that Cyt-B is able to induce cytogenetic effects in CHO cells. Using immunofluorescence microscopy, after reaction with CREST antikinetochore antibodies, we found that in cells treated with Cyt-B or Cyt-B plus MMC the frequency of MN without kinetochore was, respectively, about 70 and 85%, indicating that under our experimental conditions MN originate mainly from acentric chromatid fragments. Present data suggest that the method based on the blockage of cytokinesis by Cyt-B normally used in the MN assay should be reconsidered. Copyright 1995 S. Karger AG, Basel

Journal Article↗

PubMed1984

The murine Ah locus. Comparison of the complete cytochrome P1-450 and P3-450 cDNA nucleotide and amino acid sequences.

Mouse cytochrome P1-450 and P3-450 are most closely associated with induced aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.1) and acetanilide 4-hydroxylase activity, respectively. Full-length cDNA clones of P1-450 and P3-450 were generated from mRNA isolated from 3-methylcholanthrene-treated C57BL/6N mouse liver. P1-450 cDNA is 2620 nucleotides in length and has a coding region (base 110 to 1,675) that produces a protein with 521 residues (Mr = 58,914). P3-450 cDNA is 1,894 nucleotides in length and yields a protein with 513 residues (Mr = 58,183). P1-450 mRNA is the first reported example in mouse in which UAG is used as the termination codon. P1-450 and P3-450, both induced by polycyclic hydrocarbons and regulated by the Ah receptor, exhibit overall nucleotide and protein homology of 68, and 73%, respectively. Segments of high homology, interspersed with regions of low homology, support the hypothesis of gene conversion or unequal crossing over as possible mechanisms for divergence of these two genes. Mouse P1-450 and P3-450 cDNAs were compared with previously published data on rat P-450e cDNA and rabbit form 2 protein, corresponding to two P-450 genes from the "phenobarbital inducible" P-450 gene subfamily. Nucleotide homology between a member of either gene subfamily is about 30%, and protein homology is about 15%, suggesting that the Ah locus-associated P-450 gene subfamily diverged from the phenobarbital inducible P-450 subfamily more than 200 million years ago. An N-terminal and a C-terminal cysteinyl fragment corresponding to the regions around P1-450 Cys-158 and Cys-458, respectively, are the only two cysteinyl peptides conserved among all four proteins compared. Because of greater homology in the C-terminal conserved cysteinyl fragment between the two gene subfamilies and a greater hydrophobic pocket in the C-terminal conserved cysteinyl fragment, the data favor this cysteine as the more likely candidate for the thiolate ligand to the heme iron in the P-450 enzyme active-site.

Amino Acid Sequence↗

PubMed1996

Expression of hsp70 mRNA is induced in the brain of transgenic mice overexpressing human CuZn-superoxide dismutase following transient global cerebral ischemia.

We examined hsp70 mRNA expression in the brains of transgenic (Tg) mice overexpressing CuZn-superoxide dismutase and in wild-type (Wt) littermates after 3 min of bilateral common carotid artery occlusion. Significant induction of hsp70 mRNA occurred in the hippocampus, the cortex, and other brain regions of the Tg mice 4 h after ischemia compared to the Wt mice. However, there was no histological damage in the brains of Tg and Wt mice as assessed by both Cresyl violet staining and by TUNEL staining for DNA fragmentation. These data suggest that high levels of CuZn-superoxide dismutase activity increase hsp70 mRNA expression and that intense hsp70 mRNA expression does not predict neuronal damage, even in vulnerable hippocampal CA1 neurons.

Animals↗